Methods and devices for stimulating an immune response using nanosecond pulsed electric fields

ABSTRACT

Nanosecond pulsed electric field (nsPEF) treatments of a tumor are adjusted based on a size and type of the tumor to stimulate an immune response against the tumor and other tumors in the subject. Calreticulin expression on tumor cells can be detected to confirm treatment. An immune response biomarker can be measured, and further nsPEF treatments can be performed if needed to stimulate or further stimulate the immune response. Cancers that have metastasized may be treated by directly treating a tumor that is most accessible. The treatment can be combined with CD47-blocking antibodies, doxorubicin, CTLA-4-blocking antibodies, and/or PD-1-blocking antibodies. Electrical characteristics of nsPEF treatments can be based on the size, type, and/or strength of tumors and/or a quantity of tumors in the subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S.application Ser. No. 15/873,810, filed Jan. 17, 2018, which is acontinuation application of U.S. application Ser. No. 15/484,550, filedApr. 11, 2017, which is a divisional application of U.S. applicationSer. No. 14/805,828, filed Jul. 22, 2015 (issued as U.S. Pat. No.9,656,066), which is a divisional application of U.S. application Ser.No. 14/287,957, filed May 27, 2014 (issued as U.S. Pat. No. 9,101,764),which claims the benefit of U.S. Provisional Application No. 61/830,564,filed Jun. 3, 2013, which are hereby incorporated by reference in theirentireties for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under award numbersR01CA125722 and R44CA150484 awarded by the National Institutes of Health(NIH). The government has certain rights in the invention.

FIELD

The present application generally relates to surgical devices andmethods of use, specifically those involving nanosecond pulsed electricfields (nsPEF).

BACKGROUND Description of the Related Art

Melanoma affecting the skin is one of the top six cancers in the UnitedStates, and its rate is rising in some populations. Ultravioletradiation from sun exposure is a leading cause of such skin cancer.Cutaneous melanoma causes a skin tumor or other lesion, which isnormally treated by surgical removal.

Surgical excision of a tumor can result in an infection and leave ascar. Furthermore, if there are more tumors, every cancerous tumorshould be identified and individually excised by a surgeon. This can betime consuming and expensive, not to mention uncomfortable for patients.

Cancerous tumors that are internal to a patient may be especiallydifficult to remove, let alone detect and treat. Many patients' livesare turned upside down by the discovery of cancer in their bodies,sometimes which have formed relatively large tumors before beingdetected.

A “nanosecond pulsed electric field,” sometimes abbreviated as nsPEF,includes an electric field with a pulse width of between 0.1 nanoseconds(ns) and 1000 nanoseconds, or as otherwise known in the art. It issometimes referred to as sub-microsecond pulsed electric field. NsPEFsoften have high peak voltages, such as 10 kilovolts per centimeter(kV/cm), 20 kV/cm, to 500 kV/cm. Treatment of biological cells withnsPEF often uses a multitude of periodic pulses at a frequency rangingfrom 0.1 per second (Hz) to 10,000 Hz.

NsPEFs have been found to trigger both necrosis and apoptosis incancerous tumors. Selective treatment of such tumors with nsPEFs caninduce apoptosis within the tumor cells without substantially affectingnormal cells in the surrounding tissue due to its non-thermal nature.

An example of nsPEF applied to biological cells is shown and describedin U.S. Pat. No. 6,326,177 (to Schoenbach et al.), which is incorporatedherein by reference in its entirety for all purposes.

The use of nsPEF for the treatment of tumors is a relatively new field.There exists a need in the art for the safe and effective treatment ofcancer in human subjects.

BRIEF SUMMARY

Generally, stimulating an immune response of a human being or othersubject using nanosecond pulsed electric field (nsPEF) treatments of atumor to trigger immunogenic apoptosis in the tumor, and verifying thatthe immune response in the subject has been stimulated, is described.Based on a measurement of the immune response (or lack thereof), afurther nsPEF treatment on the same or another tumor can be implemented.Electrical characteristics of nsPEF treatments can be based on the sizeand type of tumor, as well as the degree of tumor sensitivity to nsPEFand/or a quantity of tumors in the subject.

Sufficient treatment of a cancerous tumor by nsPEF can cause theexpression of calreticulin (CRT) on the external surface membranes ofits tumor cells. Calreticulin expression may be optimized to trigger theimmune response against the underlying cancer. CD47 (cluster ofdifferentiation 47)-blocking antibodies can be injected into the subjectin conjunction with nsPEF treatment of a tumor as a combination therapy.

Not only can the triggered immune response attack the nsPEF-treatedtumor, but it can also attack like-cancer cells in other tumorsthroughout the subject's body where a cancer has metastasized.Therefore, not every tumor in the body must be individually subject tonsPEF treatments. Instead, a readily accessible, electrode-compatibletumor may be treated, and then other tumors can be monitored forshrinkage.

Metastasized cancer can also be treated by extracting circulating tumorcells (CTCs) from the subject's bloodstream, subjecting them to nsPEFsufficient to cause calreticulin expression, and then reinjecting theminto the patient's bloodstream. In some cases, a tumor may be removedfrom a patient, treated by nsPEF sufficient to cause calreticulinexpression, and then reimplanted back into a patient's body. Thereinjected CTCs or reimplanted tumor can then trigger an immune responseagainst the cancer.

Some embodiments of the present invention are related to a method forstimulating an immune response to a disease in a subject. The methodincludes positioning a set of electrodes in proximity to a tumor of adisease of a subject, applying, using the electrodes, sub-microsecondpulsed electric fields to the tumor sufficient to cause the tumor toexpress calreticulin on surface membranes of tumor cells of the tumor,and then measuring an immune response biomarker in a sample of thesubject in order to confirm a stimulation of an immune response of thesubject against the disease.

The method can be performed on human subjects as well as other mammaland animal subjects. The disease can be cancerous or noncancerous.

The method can include further applying of sub-microsecond pulsedelectric fields sufficient to stimulate immunogenic apoptosis in thetumor, and it can expressly include detecting calreticulin on surfacemembranes of the tumor cells after the applying.

The method can include introducing CD47-blocking antibodies into thesubject, the CD47-blocking antibodies neutralizing CD47 on the surfacemembranes of the tumor cells whilst the calreticulin is expressed on thesurface membranes. Introducing of CD47-blocking antibodies can occurbefore the positioning or the applying of sub-microsecond pulsedelectric fields.

The method can include introducing injecting doxorubicin, CTLA-4(cytotoxic T-lymphocyte antigen 4)-blocking antibodies, and/or PD-1(programmed death 1)-blocking antibodies into the subject before theapplying.

The measuring of the immune response biomarker can include measuring aconcentration or level of white blood cells in the sample, such as CD4+or CD8+T lymphocytes. Measuring the immune response biomarker caninclude measuring a concentration or level in the sample of a memberselected from a group consisting of white blood cells, inflammatorycytokines, C-reactive proteins, and antibodies of cancer cell markers.The method can further include gauging the immune response biomarker ina sample of the subject before the applying, and comparing results ofthe gauging of the immune response biomarker and results of themeasuring of the immune response biomarker in order to confirm thestimulation of the immune response of the subject.

The method can include administering an immune booster to the subjectwithin fourteen days of the applying. It can also include preventing,averting, or forestalling chemotherapy treatment within one month afterthe applying.

The method can include monitoring the immune response biomarker overtime in samples from the subject, perhaps measuring the immune responsebiomarker between fourteen to twenty-eight days after the applying. Onecan treat the tumor again based on the monitoring by positioning a setof electrodes in proximity to the tumor of the subject, and applyingnanosecond pulsed electric fields to the tumor sufficient to cause thetumor to express calreticulin on surface membranes of tumor cells of thetumor and sufficient to stimulate apoptosis in the tumor. The method caninclude treating a second tumor in the subject, based on the monitoring,by positioning a set of electrodes in proximity to the second tumor ofthe subject, and applying nanosecond pulsed electric fields to thesecond tumor sufficient to cause the tumor to express calreticulin onsurface membranes of tumor cells of the second tumor and sufficient tostimulate apoptosis in the second tumor.

The sub-microsecond pulsed electric fields can have pulse lengths ofbetween 0.1 and 1000 nanoseconds. The sub-microsecond pulsed electricfields can have pulse lengths of between 10 and 900 nanoseconds. Thesub-microsecond pulsed electric fields can have pulse lengths of about100 nanoseconds. The sub-microsecond pulsed electric fields can havepulse amplitudes of at least 20 kilovolts per centimeter.

The method can include determining a size of the tumor, determining atype of the tumor, and applying a number of pulses greater than 50 basedon the determined size and type of the tumor. The method can includecalculating a target treatment energy based on the determined size andtype of the tumor, and selecting a number of pulses greater than 50, anamplitude of at least 20 kilovolts per centimeter, or a pulse lengthbetween 0.1 and 1000 nanoseconds for the nanosecond pulsed electricfields based on the calculated target treatment energy.

A machine-readable tangible storage medium embodying informationindicative of instructions for causing one or more machines to performthe calculating and selecting operations. The method can further includeselecting a repetition frequency of the nanosecond pulsed electricfields based on the determined size and type of the tumor.

The method can include quantifying a strength (e.g., a resistance of atumor in response to a certain treatment, quantifying nsPEF pulseparameters required to cause apoptosis in the tumor) of tumor cells ofthe tumor, and applying a number of pulses greater than 50 based on thequantifying. The electrodes can include a pair of electrodes, and thepositioning includes positioning one of the pair of electrodes on oneside of the tumor and the other of the pair of electrodes on an opposingside of the tumor, thereby causing the electric fields to pass throughthe tumor.

The electrodes can include configurations selected from the groupconsisting of parallel plate electrodes, hemicircular electrodes,six-pole dual electrodes, and two- to fourteen-needle electrodesarranged in two parallel linear arrays.

Some embodiments are related to a method for stimulating an immuneresponse to a tumor in a subject. The method includes gauging an immuneresponse biomarker in a sample of a subject, identifying a size and atype of a tumor in the subject, calculating a target treatment energybased on the size and type of the tumor, selecting a number of pulsesgreater than 50, an amplitude of at least 20 kilovolts per centimeter,and/or a pulse length of between 0.1 and 1000 nanoseconds forsub-microsecond pulsed electric fields based on the calculated targettreatment energy, flanking at least one pair of electrodes in or aroundthe tumor or a portion thereof, applying to the tumor, using the atleast one pair of electrodes, sub-microsecond pulsed electric fieldshaving the selected number of pulses, selected amplitude, or selectedpulse length, and then waiting at least seven days, and then measuringthe immune response biomarker in a sample from the subject, comparingthe measured and gauged immune response biomarkers, adjusting the targettreatment energy based on the comparison, determining a number of pulsesgreater than 50, an amplitude of at least 20 kilovolts per centimeter,and/or a pulse length of between 0.1 and 1000 nanoseconds for nanosecondpulsed electric fields based on the adjusted target treatment energy;and treating the tumor again by flanking at least one pair of electrodesaround the tumor, and applying to the tumor sub-microsecond pulsedelectric fields based on the determined number of pulses, determinedamplitude, or determined pulse length.

The method can be performed on human subjects as well as other mammaland animal subjects. The disease can be cancerous or noncancerous.

Some embodiments are related to a method of reducing metastasis of adisease in a subject. The method includes determining locations formultiple tumors in a subject, measuring a size of each tumor, selectingone of multiple tumors based on an accessibility of the locations andmeasured sizes of the tumors, and applying, using electrodes,sub-microsecond pulsed electric fields to the selected tumor sufficientto cause the tumor to express calreticulin on surface membranes of tumorcells of the selected tumor.

The method can be performed on human subjects as well as other mammaland animal subjects. The disease can be cancerous or noncancerous.

The accessibility of each tumor can be determined by identifyingpathways to opposed sides of the respective tumor that can be contactedby electrodes through pierceable paths in the subject. A tumor can beselected based on the selected tumor being adjacent to a stomach wall.

Some embodiments are related to a method of reducing metastasis of acancerous disease in a subject. The method can include isolatingcirculating tumor cells (CTCs) from a bloodstream of a subject, amassingthe tumor cells into an in vitro mass of tumor cells, passingsub-microsecond pulsed electric fields through the in vitro mass oftumor cells, thereby treating the tumor cells, and then introducing thetreated tumor cells into the subject.

The method can be performed on human subjects as well as other mammaland animal subjects. The disease can be cancerous or noncancerous.

The passing of sub-microsecond pulsed electric fields can be sufficientto cause at least some of the tumor cells of the in vitro mass toexpress calreticulin on surface membranes of the at least some of thetumor cells. The method can include introducing CD47-blocking antibodiesinto the subject. The method can optionally include contacting anelectrode to the in vitro mass.

Some embodiments are related to using a biopsy from a tumor to treat acancerous disease in a subject. The method includes extracting a sampleof cells from a tumor of a subject, passing sub-microsecond pulsedelectric fields through the extracted sample of tumor cells sufficientto stimulate apoptosis in the tumor cells, thereby treating the tumorcells, confirming that the tumor cells of the sample have initiatedapoptosis and are no longer capable of dividing, and then introducingthe treated tumor cells into the subject. The removal of tumor cells canbe performed by fine needle aspiration or other suitable process.

The method can be performed on human subjects as well as other mammaland animal subjects. The disease can be cancerous or noncancerous.

Yet other embodiments relate to systems and machine-readable tangiblestorage media that employ or store instructions for the methodsdescribed above.

Reference to the remaining portions of the specification, including thedrawings and claims, will realize other features and advantages of thepresent invention. Further features and advantages of the presentinvention, as well as the structure and operation of various embodimentsof the present invention, are described in detail below with respect tothe accompanying drawings. In the drawings, like reference numbersindicate identical or functionally similar elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a nanosecond pulse generator apparatus in accordancewith an embodiment.

FIG. 2 illustrates oscilloscope traces of a pulse profile for bothvoltage and current in accordance with an embodiment.

FIG. 3 is an electrical schematic of a pulse generator shown in FIG. 1.

FIG. 4 illustrates a perspective view of a seven-needle electrode inaccordance with an embodiment.

FIG. 5A illustrates a perspective view of a two-pole electrode inaccordance with an embodiment.

FIG. 5B illustrates an end view of the electrode of FIG. 5A.

FIG. 6A illustrates a perspective view of a six-pole electrode inaccordance with an embodiment.

FIG. 6B illustrates an end view of the electrode of FIG. 6A.

FIG. 7A illustrates a perspective view of a hemispherical electrode inaccordance with an embodiment.

FIG. 7B illustrates an end view of the electrode of FIG. 7A.

FIG. 8A illustrates a perspective view of a parallel plate electrode inaccordance with an embodiment.

FIG. 8B illustrates an end view of the electrode of FIG. 8A.

FIG. 9 includes pictures of typical growth of a first melanoma injectedinto immunodeficient mice (Nu/Nu) and immunocompetent mice (SKH-1).

FIG. 10 includes pictures of a second tumor growing from cells injected14 days after the first tumor was removed by surgical excision of theprior art.

FIG. 11 includes pictures of a second tumor growing from cells injected28 days after the first tumor was removed by surgical excision of theprior art.

FIG. 12 includes pictures of a second tumor growing from cells injected14 days after the first tumor was treated with nsPEF nanoelectroablationin accordance with an embodiment.

FIG. 13 includes pictures of a second tumor growing from cells injected28 days after the first tumor was treated with nsPEF nanoelectroablationin accordance with an embodiment.

FIG. 14 is a line graph of tumor size versus time for immunosuppressed(Nu/Nu) mice in which the second tumor was injected 14 days aftersurgical removal or nanoelectroablation in accordance with anembodiment.

FIG. 15 is a line graph of tumor size versus time for immunosuppressed(Nu/Nu) mice in which the second tumor was injected 28 days aftersurgical removal or nanoelectroablation in accordance with anembodiment.

FIG. 16 is a line graph of tumor size versus time for immunocompetent(SKH-1) mice in which the second tumor was injected 14 days aftersurgical removal or nanoelectroablation in accordance with anembodiment.

FIG. 17 is a line graph of tumor size versus time for immunocompetent(SKH-1) mice in which the second tumor was injected 28 days aftersurgical removal or nanoelectroablation in accordance with anembodiment.

FIG. 18 is a graph comparing empirical results of secondary tumor growthbetween mice having surgically removed primary tumors (solid lines) andone nsPEF-treated primary tumor (dashed lines) in accordance with anembodiment.

FIG. 19 is a graph comparing empirical results of secondary tumor growthbetween mice having surgically removed primary tumors (solid lines) andthree nsPEF-treated primary tumors (dashed lines) in accordance with anembodiment.

FIG. 20 is a histological section within a secondary tumor stained toshow CD8+ cytotoxic T cells.

FIG. 21A is an image of an unstained, unbleached tumor section fixed 12days after nsPEF treatment in accordance with an embodiment.

FIG. 21B is an image of the same region as in FIG. 21A but withfluorescent labeling.

FIG. 22A is an image of an unstained, unbleached tumor section fixed 19days after nsPEF treatment in accordance with an embodiment.

FIG. 22B is an image of the same region as in FIG. 22A but withfluorescent labeling.

FIG. 23A is an image of an unstained, unbleached second tumor sectionfixed 19 days after nsPEF treatment of a first tumor in accordance withan embodiment.

FIG. 23B is an image of the same region as in FIG. 23A but withfluorescent labeling.

FIG. 24A is an image of an unstained, unbleached second tumor sectionfixed 32 days after nsPEF treatment of a first tumor in accordance withan embodiment.

FIG. 24B is an image of the same region as in FIG. 24A but withfluorescent labeling.

FIGS. 25A-25F are images of six different human pancreatic carcinomacells with calreticulin labeling after nsPEF treatment in accordancewith an embodiment.

FIGS. 26A-26F are images of six different murine (mouse) squamous cellcarcinoma cells with calreticulin labeling after nsPEF treatment inaccordance with an embodiment.

FIG. 27 is a graph showing percentages of cells that expresscalreticulin on their surface membranes after nsPEF treatment inaccordance with an embodiment.

FIG. 28A is an image of a primary liver tumor one week after injection.

FIG. 28B is an image of a secondary liver tumor three weeks after nsPEFtreatment of the first tumor (in FIG. 28A) in accordance with anembodiment.

FIG. 29A is an image of a primary liver tumor one week after injection.

FIG. 29B is an image of a secondary liver tumor one week after injectionof tumor cells that took place three weeks after nsPEF treatment of thefirst tumor (in FIG. 29A) in accordance with an embodiment.

DETAILED DESCRIPTION

It has been shown that nsPEF treatments can be used to cause canceroustumors to express calreticulin on their cell surface membranes, whichmay be sufficient to stimulate an immune response that inhibitssubsequent tumor growth and/or mitigate metastasis. An nsPEF treatmentcan be sufficient to stimulate immunogenic apoptosis in the primarytumor that is actually subject to the nsPEF pulses, while the immuneresponse can attack secondary tumors that were not so directly treated.

A “tumor” includes any neoplasm or abnormal, unwanted growth of tissueon or within a subject, or as otherwise known in the art. A tumor caninclude a collection of one or more cells exhibiting abnormal growth.There are many types of tumors. A malignant tumor is cancerous, apre-malignant tumor is precancerous, and a benign tumor is noncancerous.Examples of tumors include a benign prostatic hyperplasia (BPH), uterinefibroid, pancreatic carcinoma, liver carcinoma, kidney carcinoma, coloncarcinoma, pre-basal cell carcinoma, and tissue associated withBarrett's esophagus.

A “disease” includes any abnormal condition in or on a subject that isassociated with abnormal, uncontrolled growths of tissue, includingthose that are cancerous, precancerous, and benign, or other diseases asknown in the art.

“Apoptosis” of a tumor or cell includes an orderly, programmed celldeath, or as otherwise known in the art.

“Immunogenic apoptosis” of a tumor or cell includes a programmed celldeath that is followed by an immune system response, or as otherwiseknown in the art. The immune system response is thought to be engagedwhen the apoptotic cells express calreticulin or another antigen ontheir surfaces, which stimulates dendritic cells to engulf, consume, orotherwise commit phagocytosis of the targeted cells leading to theconsequent activation of a specific T cell response against the targettumor or cell.

Pulse lengths of between 10 and 900 nanoseconds for nsPEF have beenparticularly studied to be effective in stimulating an immune response.Pulse lengths of about 100 nanoseconds are of particular interest inthat they are long enough to carry sufficient energy to be effective atlow pulse numbers but short enough to be effective in the mannerdesired.

A time of “about” a certain number of nanoseconds includes times withina tolerance of ±1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25% or otherpercentages, or fixed tolerances, such as ±0.1, ±0.2, ±0.3, ±0.4, ±0.5,±0.7, ±1.0, ±2.0, ±3.0, ±4.0±5.0, ±7.0, ±10, ±15, ±20, ±25, ±30, ±40,±50, ±75 ns, or other tolerances as acceptable in the art in conformancewith the effectivity of the time period.

Immune system biomarkers can be measured before and/or after nsPEFtreatment in order to confirm that the immune response has beentriggered in a patient. Further, nsPEF treatment can be paired withCD47-blocking antibody treatment to better train CD8+ T cells (i.e.,cytotoxic T cells) for attacking the cancer.

U.S. Patent Application Publication No. US 2011/0288545 A1 (to Beebe etal.) discloses that mice with subdermal injections of hepatocellularcarcinoma cells (HCC) developed immunity against HCC after one nsPEFtreatment. This conclusion is based on the stagnation of secondarytumors of HCC, which were initiated in the mice after their first tumorshad been eliminated by nsPEF. Yet, there is no disclosure in thereference of measuring immune response biomarkers. Instead it wasobserved that secondary tumors injected after treatment did not grow,which is an indirect indication of an immune response. The effects ofnsPEF on concurrent secondary tumors (i.e., tumors that exist in thesubject that are not treated with nsPEF while a primary tumor is treatedwith nsPEF) are not disclosed.

In an embodiment, immune response biomarkers, such as a concentration ofCD4+ and/or CD8+T lymphocyte white blood cells in the bloodstream of apatient, are sampled in order to confirm stimulation of the patient'simmune system. Other biomarkers include concentrations and/or levels ina sample inflammatory cytokines, C-reactive proteins, and antibodies ofcancer cell markers. The biomarkers can be gauged or measured bothbefore and after nsPEF treatment in order to determine or confirmwhether, and to what extent, the subject's immune response has beenstimulated.

Combination Therapies

Induction of tumor antigen-specific cytotoxic T lymphocytes oftenresults in growth inhibition and even shrinkage of solid tumors.Presentation of tumor-derived antigens by dendritic cells (DC) might bea necessary step in the induction of an immune response to the tumor. Itis thought that DCs can only acquire antigens from apoptotic cells andstimulate antigen-specific MHC class I-restricted cytotoxic Tlymphocytes.

There are at least three factors that can influence DC phagocytosis oftumor cells: 1) Apoptotic death may be a requirement for antigenpresentation because antigens from necrotic cells cannot enter thispathway. Moreover, a high ratio of apoptotic cells-to-DCs can alsoinduce DC maturation and enhance the DC ability to efficiently presentantigens derived from the apoptotic cells to T cells. 2) Exposure ofcalreticulin on the cell surface may be an “eat me” signal thatstimulates phagocytosis of the tumor cells by DCs. 3) CD47 is commonlyhighly expressed on many tumor cells and functions as a “don't eat me”signal, inhibiting the phagocytosis by DCs. Consequently, blocking CD47using antibodies directed to it may enhance phagocytosis and lead to astronger immune response.

Proper nanoelectroablation can result in the activation of two of thesefactors for phagocytosis of the tumor cells. It triggers apoptosis andthe exposure of calreticulin on the tumor cell surface. This therapymight be even more effective if it is combined with other therapiesknown to influence one or more of the three critical factors above. Onesuch combination therapy is to administer by intraperitoneal (IP)injection antibodies to CD47 approximately twenty-four hours beforetreating with nsPEF. These antibodies can bind to tumor cell CD47 andreduce the CD47 inhibitory effect on DCs. NsPEF-stimulated calreticulinexpression can then stimulate DC phagocytosis of the tumors cells sothat the DCs can present tumor antigens to stimulate antigen-specificMHC class I-restricted cytotoxic T lymphocytes.

Another possible combination therapy is to pretreat the patient withchemotherapeutic drugs that have been found to stimulate the surfaceexpression of calreticulin. Anthracyclines and oxaliplatin are importantdrugs used in the management of leukemia, lymphoma, sarcoma and uterine,ovarian and breast cancers. They may induce immunogenic apoptosis thatis characterized by the exposure of calreticulin on the cell surface andsecretion of adenosine triphosphate (ATP). Calreticulin and ATP interactwith surface receptors on dendritic cells to promote engulfment of dyingcells and presentation of tumor antigens. One of the most popular of theanthracyclines is doxorubicin (e.g., a ADRIAMYCIN PFS® or ADRIAMYCINRDF® pharmaceutical preparation) because of its lower toxicity and highefficacy against solid tumors (Cancer Res. 71:4809, Jul. 15, 2011).Therefore, one combination therapy that can enhance the immune responseis to treat patients first with doxorubicin followed by nsPEF treatmentof the tumor.

Another combination therapy that can be used involves the T cellsthemselves. On the surface of just about every helper T cell is aprotein called either CTLA-4 or CD152. The activation of this protein onthe helper T cell inhibits the cytotoxic T cell attack when bound by atumor cell. Antibodies directed against CTLA-4 have been shown toenhance the immune response to some tumor types such a melanoma.Injecting antibodies to CTLA-4 into the patient about 24 hours beforetreating the tumor with nsPEF may achieve an enhanced immune response.

Another member of the CTLA-4 family is the “programmed death 1” (PD-1)membrane protein. It is found on the surfaces of activated T cells, Bcells, and macrophages, and it negatively regulates immune responses. Areceptor for PD-1 is PD-L1, and PD-L1 is expressed on almost all murinetumor cell lines. When PD-1 binds to this receptor, normal T cellactivation and expansion is inhibited. Antibodies developed against PD-1have been found to enhance immune function, similar to antibodiesagainst CTLA-4. Another combination therapy is to inject anti-PD-1antibodies about twenty-four hours before treating the tumor with nsPEF.

FIG. 1 illustrates a nanosecond pulse generator apparatus in accordancewith an embodiment. Pulse widths, duty cycles, and other pulseparameters are controlled by a spark gap, the critical distance of whichis controlled by compressed gas, such as compressed carbon dioxide.NsPEF system 100 includes pressure readout 101, digitizing oscilloscope102, emergency off button 103, and microcontroller interface 104, allconnected to nsPEF generation system 105 within a metal-shieldedcabinet.

A human operator inputs a number of pulses, amplitude, and frequencyinto a numeric keypad of microcontroller interface 104. In thisembodiment, the pulse width is fixed. Microcontroller sends signals to ahigh voltage power supply (HVPS) and pressure system to control a sparkgap (switch) within cabinet 105. Fiber optic cables electrically isolatethe contents of the metal cabinet with nsPEF generation system 105, thehigh voltage circuit, from the outside. In order to further isolate thesystem, system 100 is battery powered instead of from a wall outlet.

FIG. 2 illustrates oscilloscope traces of a pulse profile for bothvoltage and current in accordance with an embodiment. Output from thespark gap is shown with voltage on the top of the figure and amperage onthe bottom for a single pulse. The pulse has an amplitude of about 12 kVand an amperage of about 60 A, which lasts for approximately 100 ns.Thus, twelve kilovolts was applied to suction electrodes with 4 mmbetween the plates so that the tumors experienced 30 kV/cm, and currentvaried between 12 and 60 A. Given a voltage, current depends heavily onthe electrode type and skin resistance.

FIG. 3 is an electrical schematic of a pulse generator inside nsPEFsystem 100 wired in a transmission line arrangement (see FIG. 1). Highvoltage circuit 300 can be entirely shielded within the cabinet of nsPEFgeneration system 105. High voltage circuit 300 includes a high voltagepower supply HVPS, diode D2, resistor Rdischarge, and spark gap switch,all connected in parallel to ground. Between D2 and Rdischarge is diodeD1, and between Rdischarge and the switch is resistor Rcharge.

The switch is directly connected to the positively-charged (+) terminalsof twelve high voltage capacitors, C1 through C12. Between eachcapacitor at the other end (i.e., the opposite terminal) is ahigh-current capable inductor, L2 through L12, which are all connectedto ground in series through high current-capable inductor L1 andresistor Rtermination. Output from bank of capacitors flows to a load,modeled as an ideal resistor LOAD, to ground.

To charge the capacitors, the spark gap switch is opened and the HVPScharges capacitors C1-C12. When the spark gap switch is closed, thecapacitors rapidly discharge from their positive terminals through theswitch to ground. Electrons from the negative terminals of thecapacitors rush through inductors L1-L12 through LOAD to ground.

In an exemplary embodiment, discharging through a triggered spark gapdelivers a 100-ns long pulse with a 20 ns rise time. Microcontrollerinterface 104 is used to trigger the spark gap at 2, 3, 4, 5, or 7pulses per second (pps), control the voltage level of the power supplyand count the pulses. The pulse counter utilizes the signal generated bya custom current sensor placed around one of the wires connected to thesuction electrode so that only pulses resulting in current delivery tothe tumor are counted.

FIG. 4 illustrates a perspective view of a seven-needle suctionelectrode in accordance with an embodiment. In electrode 400, sheath 401surrounds seven sharp electrodes 402 with an broad opening at a distalend. When the open end is placed against a tumor, air is evacuated fromthe resulting chamber sufficient to draw the entire tumor or a portionthereof into the chamber. The tumor is drawn so that one or more of theelectrodes preferably penetrates the tumor. Sharp ends of the electrodesare configured to pierce the tumor. The center electrode is at onepolarity, and the outer six electrodes are at the opposite polarity.Nanopulses electric fields can then be precisely applied to the tumorusing nsPEF system 100 (see FIG. 1).

The electrodes can be opposed, one of each positive and negative pair ofelectrodes on one side of a tumor and the other electrode of the pair onan opposing side of the tumor. Opposing sides of a tumor can includeareas outside or within a tumor, such as if a needle electrode pierces aportion of the tumor.

FIGS. 5A-5B illustrate a two-pole suction electrode in accordance withan embodiment. In electrode device 500, sheath 501 surrounds two broadelectrodes on opposite sides of a chamber. When air is evacuated and atumor is pulled within the chamber, the opposing electrodes apply nsPEFpulses to the tumor.

FIGS. 6A-6B illustrate a six-pole suction electrode in accordance withan embodiment. In electrode device 600, sheath 601 surrounds sixelectrodes 602, which are spaced equally around the circumference of thechamber. One or more of electrodes 602 can be energized in order toeffectively pulse a tumor, which is drawn inside by suction.

FIGS. 7A-7B illustrate a hemispherical suction electrode in accordancewith an embodiment. In electrode device 700, sheath 701 surrounds twoelectrodes 702 that curve around a majority of the circumference of thechamber. Relatively small gaps separate electrodes 702.

FIGS. 8A-8B illustrate a parallel plate suction electrode in accordancewith an embodiment. In electrode device 800, circular cross-sectionsheath 801 surrounds a rectangular chamber. Along the long sides of therectangles, flat electrodes 802 are opposed from one another.

The nature of the electrode used mainly depends upon the shape of thetumor. Its physical size and stiffness can also be taken into account inselection of a particular electrode type.

U.S. Pat. No. 8,688,227 B2 (to Nuccitelli et al.) discloses othersuction electrode-based medical instruments and systems for therapeuticelectrotherapy, and it is hereby incorporated by reference.

If there are multiple tumors in a subject, a surgeon can select a singletumor to treat based on the tumors compatibility with electrodes. Forexample, a tumor that is adjacent to a stomach wall may be more easilyaccessible than one adjacent a spine or the brain. Because a nsPEF pulseis preferably applied so that the electric field transits through asmuch tumor mass as possible while minimizing the mass of non-tumor cellsthat are affected, a clear path to two opposed ‘poles’ of a tumor mayalso be a selection criterion.

A “pierceable path” is a pathway that is unobstructed by bone, nervoussystem conduits, vital organs, or other material that is eitherdifficult to pierce with an electrode or more sensitive to damage thanother regions of the body.

For tumors on or just underneath the skin of subject, needle electrodescan be used percutaneously. For locations deeper within a subject, aretractable electrode can fit into a gastroscope, bronchoscope,colonoscope, or other endoscope or laparoscope. For example, a tumor ina patient's colon can be accessed and treated using an electrode withina colonoscope. When moving into position within the body of the subject,the retractable electrode is in a retracted position; when in positionat a tumor, the retractable electrode is deployed.

Barrett's esophagus, in which portions of tissue lining a patient'sesophagus are damaged, may be treated using an electrode placed on aninflatable balloon.

FIG. 9 includes pictures of typical growth of a first melanoma injectedinto immunodeficient mice (Nu/Nu) and immunocompetent mice (SKH-1). Theleftmost column shows reflected light surface views, the second columnshows transilluminated views, and the third column shows fluorescentimages of the Nu/Nu mice. The fourth column shows reflected lightsurface views, the fifth column shows transilluminated views, and therightmost column shows fluorescent images of the SKH-1 mice. The dayafter injection on which the photographs were taken is indicated in theupper left of the reflected light image.

The photographs show normal (control) growth of a melanoma after 2 and 9days. One can see that the tumor enlarges.

FIG. 10 includes pictures of a second tumor growing from cells injected14 days after the first tumor was removed by surgical excision of theprior art. The arrangement of pictures is the same of that as in FIG. 9.

The photographs show normal (control) growth of second tumors. One cansee that the tumors grow essentially the same as the first tumors.

FIG. 11 includes pictures of a second tumor growing from cells injected28 days after the first tumor was removed by surgical excision of theprior art. The arrangement of pictures is the same of that as in FIG. 9.

The photographs show normal (control) growth of second tumors. One cansee that the tumors grow essentially the same as the first tumors.

FIG. 12 includes pictures of a second tumor growing from cells injected14 days after the first tumor was treated with nanoelectroablation inaccordance with an embodiment. The arrangement of pictures is the sameof that as in FIG. 9.

The photographs show a difference in secondary tumor growths betweenimmunodeficient mice (Nu/Nu) and normal mice (SKH-1). One can see thatthe secondary tumor in the normal mice hardly grew, while the secondarytumor in the immunodeficient mice grew unabated. This demonstrates thatthe difference is caused by sufficient stimulation of the immune systemby nsPEF.

FIG. 13 includes pictures of a second tumor growing from cells injected28 days after the first tumor was treated with nanoelectroablation inaccordance with an embodiment. The arrangement of pictures is the sameof that as in FIG. 9.

The photographs show a difference in secondary tumor growths betweenimmunodeficient mice (Nu/Nu) and normal mice (SKH-1). One can see thatthe secondary tumor in the normal mice hardly grew, while the secondarytumor in the immunodeficient mice grew unabated. This demonstrates thatthe nsPEF-inspired stimulation of the immune system against secondarytumors is sufficient for tumors that are injected even after 28 days.That is, nsPEF treatment of a primary tumor stimulates the immune systemfor weeks or months on end, if not longer or permanently.

FIG. 14 is a line graph of tumor size versus time for immunosuppressed(Nu/Nu) mice in which the second tumor was injected 14 days aftersurgical removal or nanoelectroablation in accordance with anembodiment. Each mean is an average of three separate experiments, andthe bars represent SEM (standard error of mean).

Surgical removal and nsPEF does not appear to have any effect onsecondary tumors in immunosuppressed mice.

FIG. 15 is a line graph of tumor size versus time for immunosuppressed(Nu/Nu) mice in which the second tumor was injected 28 days aftersurgical removal or nanoelectroablation in accordance with anembodiment. Each mean is an average of three separate experiments, andthe bars represent SEM (standard error of mean).

Again, surgical removal and nsPEF does not appear to have any effect onsecondary tumors in immunosuppressed mice.

FIG. 16 is a line graph of tumor size versus time for immunocompetent(SKH-1) mice in which the second tumor was injected 14 days aftersurgical removal or nanoelectroablation in accordance with anembodiment.

There is a striking difference between the growth of secondary tumors inwhich their primary tumors are subject to surgical removal or nsPEF.NsPEF of a primary tumor results in a secondary tumor shrinking in theseimmunocompetent mice.

FIG. 17 is a line graph of tumor size versus time for immunocompetent(SKH-1) mice in which the second tumor was injected 28 days aftersurgical removal or nanoelectroablation in accordance with anembodiment.

Again, there is a striking difference between secondary tumor growthrates. One secondary tumor decreased in size so substantially that it isevident on the logarithmic scale.

Immunodeficient mice showed no difference between the tumor growth ratesof the initial and secondary tumors. However, in immunocompetent mice,most tumors injected 28 days after the first tumor was treated withnsPEF actually shrank. Immunohistochemical analysis of these tumorsidentified CD4+ cells within both the treated tumor as well as inuntreated tumors in animals in which another tumor had beennanoelectroablated.

However, one drawback of this experiment was that the B16 melanoma cellswere originally derived from C57BL/6 mice so that the SKH-1 strainrecognized these B16 cells as foreign. Consequently, the controls inwhich the tumor was surgically removed rather than treated with nsPEFalso exhibited some inhibition of secondary tumor growth, although to alesser degree than that observed in mice with nsPEF-treated tumors.

In order to eliminate this complication, B6 albino mice derived from theC57BL/6 line were used for subsequent experiments. These B6 mice do notmount a measurable immune response against B16 melanoma cells;therefore, the secondary tumor grows just as fast as the primary tumor.

The inventors conducted two types of experiments with B6 albino mice. Inthe first, a single tumor was injected into several mice and eithersurgically removed or treated with nsPEF seven days post-injection whenthe tumors were 4 mm in diameter. In the second experiment, threeprimary tumors were injected and either surgically removed or treatedwith nsPEF seven days post injection. Twenty-eight days later, asecondary tumor was injected and its growth rate measured for 10-13days. No inhibition of secondary tumor growth of the tumors in mice wasobserved in which the primary tumors were surgically removed. However,in experiments in which the primary tumors were treated with nsPEF, thesecondary tumor injected 28 days later exhibited inhibited growthcompared to the controls without a second nsPEF treatment. In two of thethree treated mice, the secondary tumor actually shrank to about half ofits original size by day ten (see FIG. 19).

FIG. 18 is a graph comparing empirical results of secondary tumor growthbetween mice having surgically removed primary tumors (solid lines) andone nsPEF-treated primary tumor (dashed lines) in accordance with anembodiment. One 4 mm-wide primary tumor was either treated with nsPEF orremoved surgically followed by the injection of a second tumor 28 dayslater. Note that the dashed lines mostly fall below the solid lines,indicating that nsPEF treatments of primary tumors helps in haltinggrowth (or shrinking) secondary tumors.

FIG. 19 is a graph comparing empirical results of secondary tumor growthbetween mice having surgically removed primary tumors (solid lines) andthree nsPEF-treated primary tumors (dashed lines) in accordance with anembodiment. Three 4 mm-wide tumors were treated with nsPEF or surgicallyremoved followed by the injection of a second tumor 28 days later. Amonga three-tumor data set, a clearer trend of dashed lines below solidlines is visible. A few of the nsPEF treatments resulted in secondarytumors shrinking to half (0.5) their size.

Secondary tumor growth is inhibited in B6 albino immunocompetent mice inwhich the first tree tumors were nanoelectroblated (three dashed linedata sets marked with Xs) but not in mice in which the first threetumors are surgically removed (solid line data set marked with solidOs). The black line data set marked with diamonds represents the averagegrowth of the primary tumors prior to removal or nanoelectroablation.

FIG. 20 is a histological section of a region within a secondary tumorstained with a fluorescent antibody to label CD8+ cytotoxic T cells, aresult of nsPEF stimulation of primary tumors in accordance with anembodiment. This shows that the subject's immune system was sufficientlystimulated with nsPEF.

FIGS. 21A-21B are images of an unstained and fluorescently labeled tumorsection, respectively, fixed 12 days after nsPEF treatment. The whitescale bar represents 200 μm.

The fluorescent label from a CD4 antibody indicates surface labeling,but no T cells could be found within the tumor at this early time point.

FIGS. 22A-22B are images of an unstained and fluorescently labeled tumorsection, respectively, fixed 19 days after nsPEF treatment.

Superposition of both Hoechst and anti-CD4 fluorescence from the sectionshown indicates the presence of CD4+ T cells (arrows) within the tumorin FIG. 22B. That is, after 19 days of this primary tumor, CD4+ T cellswere activated against the tumor.

FIGS. 23A-23B are images of an unstained and fluorescently labeledsecondary tumor section, respectively, fixed 19 days after nsPEFtreatment of a primary tumor.

Superposition of Hoechst and anti-CD4 fluorescent labels from thesection shown indicates the presence of CD4+ T cells (arrows) within the(untreated) secondary tumor in FIG. 23B.

FIGS. 24A-24B are images of an unstained and fluorescently labeledsecond tumor section fixed 32 days after nsPEF treatment of a firsttumor.

Superposition of Hoechst and anti-CD4 fluorescent labels from thesection indicates the presence of CD4+ T cells (arrows) are found withinthis untreated (i.e., not directly treated by nanoelectroablation)tumor.

Each of FIGS. 21-24 illustrate the arrival of CD4+ T cells against theprimary (directly treated) and secondary (untreated) tumors, a result ofproper nsPEF stimulation.

FIGS. 25A-25F are images of six different human pancreatic carcinomacells with calreticulin labeling after nsPEF treatment in accordancewith an embodiment. Labeled with fluorescent anti-calreticulin antibody,the figures indicate the translocation of calreticulin to the respectivehuman pancreatic carcinoma cell surfaces following nsPEF stimulation.

The figures shows human pancreatic carcinoma cells that were fixed andlabeled two hours after being treated with 15 pulses of 25 kV/cm and 100ns. It does not take long for calreticulin to be detectable on thesurface membranes of the tumor cells after nsPEF treatment.

FIGS. 26A-26F are images of six different murine (mouse) squamous cellcarcinoma cells with calreticulin labeling after nsPEF treatment inaccordance with an embodiment. Labeled with fluorescentanti-calreticulin antibody, the figures indicate the translocation ofcalreticulin to the respective murine squamous cell carcinoma cellsurfaces following nsPEF stimulation.

The figures shows murine squamous cell carcinoma cells that were fixedand labeled two hours after being treated with 25 pulses of 25 kV/cm and100 ns.

Calreticulin is a protein normally found in the endoplasmic reticulum inthe cell interior. Some treatments that trigger apoptosis have beenfound to stimulate the translocation of this protein to the plasmamembrane where it serves as an additional “eat me” signal for whiteblood cells. It is a member of the Damage-Associated Molecular Pattern(DAMPs) family that has been shown to stimulate an immune responseagainst the cells exhibiting those proteins on their surface.

Such sampling of tumor cells for calreticulin can be used to ensureproper, sufficient, and optimized stimulation of the immune system. Forexample, calreticulin can be detected on 0.5%, 1%, 2%, 3%, 4%, 5%, 7%,10%, 12%, 15%, 20%, 25%, 30%, 33%, or more of the surface of a cell canindicate that it is sufficiently expressed for purposes of stimulatingan immune response. The amount of calreticulin may be chosen in order tooptimize immune response. The amount of calreticulin expression in aseries of tumor cells can be controlled by the electric field intensity(e.g., voltage per centimeter), pulse width, number of pulses, and dutycycle of the pulses of the nsPEF treatment. The electrical currentflowing through the cells may be indirectly controlled by the electricfield intensity, a current limiter, or as otherwise known in the art.

FIG. 27 is a graph showing percentages of cells that expresscalreticulin on their surface membranes after nsPEF treatment inaccordance with an embodiment. Relative percentages are shown of bothmurine squamous cell carcinoma cells and human pancreatic carcinomacells exhibiting calreticulin on their surface in response to nsPEFtreatment at various pulse numbers (i.e., 25 kV/cm, 100 ns for theexemplary embodiment). As shown for isolated human pancreatic carcinomatumors, 10 pulses maximized calreticulin expression. A maximumcalreticulin expression peak may be optimized by the number of pulses.

For murine squamous cell carcinoma cells, 25 pulses triggers CRTtranslocation in nearly 30% of the treated cells. This treatment wasdone in a cuvette with parallel plates 2 mm to 4 mm apart. The cellswere floating free in the medium rather than packed densely in a tumor.Under these conditions, the cells are more sensitive to the imposedfield and respond to much lower pulse numbers than when they are in atumor in vivo.

FIG. 28A is an image of a primary liver tumor 1 week after the injectionof tumor cells into the liver and just before it was nsPEF treated.After the FIG. 28A photograph was taken, the tumor was subject to nsPEFtreatment. After three weeks, the first tumor was completely ablated anda second injection of tumor cells was made into the same liver. One weeklater, the FIG. 28B photograph was taken of the secondary tumor. Themuch smaller size of this second tumor after the same growth period of 1week suggests that the immune system was inhibiting the growth of thesecond tumor.

To rule out that the immune system was being stimulated by the tumorcells themselves (as foreign objects to the body), syngeneic (i.e.,genetically identical) tumor cells were used. These tumors weregenerated by injecting 10⁶ McA-RH7777 rat liver tumor cells into theliver of a Buffalo rat.

FIG. 29A is an image of a primary liver tumor after one week of growth,just before it is nsPEF treated. After the FIG. 29A photograph wastaken, the tumor was subjected to nsPEF treatment. After three weeks, asecond injection of tumor cells was made in the liver. After one week ofgrowth, the FIG. 29B photograph was taken of the secondary tumor. Thegrowth of the secondary tumor was strongly inhibited, and the tumor sizeafter 1 week of growth was 95% smaller on average than the size of thefirst tumor after 1 week of growth.

The figure provides evidence that nanoelectroablation of a liver tumortriggers an immune response against the treated tumor. The secondinjection of the same number of tumor cells into a different liver loberesults in a strong inhibition of tumor growth that is probably due toan immune response as found with the second melanoma tumor injectiondescribed above.

Applying nsPEF to a tumor sufficient to stimulate apoptosis includes atleast the electrical characteristics found experimentally. For example,a 100 ns long pulse with a 20 ns rise time to 30 kV/cm (kilovolts percentimeter) at 1 to 7 pulses per second (pps) for 500 to 2000 pulses hasbeen found to be sufficient to stimulate apoptosis, depending on thetumor type. Pulsed electric fields of at least 20 kV/cm have been shownto be effective. A number of pulses greater than 50 pulses has also beenshown to be effective. Current values between 12 A and 60 A resulted,depending on the electrode type and skin resistance.

For a 2-pole parallel plate, the frequency was 7 pps, and for needleelectrodes, the frequency was lowered to 5 pps. If tumor regrowthoccurred, one or more other treatments were applied until there was nosign of recurrence.

Measuring an immune response biomarker between 14 and 28 days has beenshown to be an effective time period for determining whether thenanoelectroablation treatment successfully stimulated an immuneresponse. Gauging an immune response biomarker before treatment and thencomparing the immune response biomarker after treatment to thepre-treatment biomarker can result in a better measure of immuneresponse in some situations.

Subsequent nanoelectroablation treatment of the same or a differenttumor can re-stimulate an immune response or begin stimulating an immuneresponse if the first treatment was unsuccessful. A quantifiedsensitivity of the tumor cells to nsPEF can include a measured growth orreduction in size in a tumor at a specified time after nsPEF therapy.

Stimulation of Immune Response

It has been surprisingly found that an nsPEF that is applied to a tumorin a subject can stimulate the production of an immune response to thetumor in the subject. Without being bound to a particular theory, it isbelieved that application of the nsPEF to the tumor stimulates,promotes, or potentiates immunogenic apoptosis in the tumor cell andthat subsequently, an immune response to the tumor cells or cellfragments is stimulated. The immune response to the tumor cells inhibitssubsequent tumor growth (e.g., metastatic tumor growth). Thus,stimulating immunogenic apoptosis of tumor cells results in thestimulation of an immune response to the tumor cells that can delayand/or inhibit the growth of subsequent tumors in the subject, reducethe size of subsequent tumors in the subject, delay and/or inhibit tumormetastasis in the subject, and/or reduce the size or spread ofmetastatic tumors in the subject.

Thus, in one aspect the present invention relates to methods ofstimulating an immune response to a tumor in a subject by applying ansPEF to the tumor to stimulate apoptosis of the tumor cells andstimulating the production of an immune response to the tumor cells. Inanother aspect, the present invention relates to methods of preventingsubsequent tumor growth (e.g., metastatic tumor growth) in a subject byapplying a nsPEF to the tumor to stimulate apoptosis of the tumor andstimulating the production of an immune response to the tumor cells.

A nsPEF treatment that is sufficient to stimulate an immune response maydo so by causing the tumor to express calreticulin (CRT) on surfacemembranes of its tumor cells. CRT is a well-documented DAMP that canfunction as either adjuvant or danger signals for the innate immunesystem of a subject. Some DAMPs become enriched on the outer leaflet ofplasma membranes, such as calreticulin and HSP90. These DAMPs can have abeneficial role in cancer therapy by stimulating an immune system. Ithas been demonstrated that blockade or knockdown of calreticulinsuppresses the phagocytosis of anthracyclin-treated tumor cells bydendritic cells and abolishes their immunogenicity in mice (Obeid etal., “Calreticulin exposure dictates the immunogenicity of cancer deathcells,” Nature Medicine, January 2007, volume 13(1), pages 54-61). Ithas also been shown that the amount of phagocytosis by dendritic cellsis directly proportional to the amount of calreticulin expression oncell surfaces of a target cell. The inventors have demonstrated withtheir nsPEF equipment that it can trigger calreticulin on the cellsurface of treated cells. This expression of calreticulin on surfacemembranes of tumor cells then stimulates the subject's immune response.

Cancer that has metastasized through a subject's bloodstream may betreated using nsPEF's immune stimulation properties. For treatment,circulating tumor cells (CTCs) are isolated from the bloodstream andamassed in vial, test tube, or other suitable in vitro environment. Insome cases, there may only be a few (e.g., 5, 10), tumor cells that arecollected and amassed. Through this mass, an nsPEF electric field isapplied in order to treat the cells. This may or may not causecalreticulin to be expressed on the surface membranes of the tumorcells. The tumor cells may then be introduced back into the subject'sbloodstream by injection, infusion, or otherwise.

In an alternative embodiment, single CTCs may also be isolated from thebloodstream, and each tumor cell treated individually. An automatedsystem that captures CTCs in whole blood using iron nanoparticles coatedwith a polymer layer carrying biotin analogues and conjugated withantibodies for capturing CTCs can automatically capture the tumor cells,and a magnet and or centrifuge can separate them. After separation fromthe antibodies, the CTCs may be treated with nsPEF through a smallcapillary and then reintroduced to the patient's bloodstream.

The treated CTCs, with calreticulin expressed, can trigger an immuneresponse in the subject against the cancer. A technical advantage ofthis method is that invasive surgery to remove a tumor may be avoided bysimply treating CTCs. Further, a large number of tumors may be addressedat one time simply by triggering the body's own immune response. In vivoelectroshocks, and their associated side effects, are avoided.

In another embodiment, in vivo electroshocks and invasive surgery maywish to be avoided. A biopsy may be performed to remove a sample oftumor cells from one or a subset of multiple tumors within a patient.The sample may be cut, split from, peeled, or otherwise dissociated fromthe tumor. The sample is then subject to nsPEF treatment. Aftertreatment, an assay confirms that treated samples have been effectivelyablated, experience apoptosis so that they will no longer divide, and/orsufficiently express calreticulin on surface membranes of tumor cells ofthe sample. After confirmation, the biopsy samples are re-injected orotherwise reintroduced into the patient. A technical advantage of thisover treating CTCs is that a much larger number of cells may be treatedand reintroduced into the patient.

While examples in the application discuss human and murine subjects, thetreatment of other animals is contemplated. Agricultural animals, suchas horses and cows, or racing animals, such as horses, may be treated.Companion animals, such as cats and dogs, may find special use with thetreatments described herein. It may be difficult for a veterinarian toremove many tumors from a small animal, and cancers may be caughtrelatively late because the animals cannot communicate their advancingpain. Further, the risk inherent in reinjecting tumor cells—albeittreated tumor cells—may be worth the potential benefits of potentiallyhalting a metastasized cancer in a loved pet.

The methods of the present invention can be used for the treatment ofany type of cancer, whether characterized as malignant, benign, softtissue, or solid, and cancers of all stages and grades including pre-and post-metastatic cancers. Examples of different types of cancerinclude, but are not limited to, digestive and gastrointestinal cancerssuch as gastric cancer (e.g., stomach cancer), colorectal cancer,gastrointestinal stromal tumors, gastrointestinal carcinoid tumors,colon cancer, rectal cancer, anal cancer, bile duct cancer, smallintestine cancer, and esophageal cancer; breast cancer; lung cancer;gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer;prostate cancer, ovarian cancer; renal cancer (e.g., renal cellcarcinoma); cancer of the central nervous system; skin cancer (e.g.,melanoma); lymphomas; gliomas; choriocarcinomas; head and neck cancers;osteogenic sarcomas; and blood cancers.

Electrical characteristics of nsPEF treatments can be adjusted based ona size and/or a type of a tumor. Types of tumors may include tumors ofdifferent regions of the body, such as the cancerous tumors describedabove.

Measuring a Stimulated Immune Response

In some embodiments, the methods of the present invention compriseapplying a nanosecond pulsed electric field (nsPEF) to a tumor in asubject and measuring the stimulation of immune response to the tumor inthe subject. As used herein, the term “measuring the stimulation ofimmune response” in a subject includes determining the presence or levelof one of more biomarkers of immune response in one or more samples fromthe subject by using any quantitative or qualitative assay known to oneof skill in the art. In some embodiments, an immune response isstimulated in a subject if the level of expression of one or morebiomarkers of immune response (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore biomarkers) is above a threshold level (e.g., a threshold leveldetermined based on an average or mean level of immune response in asubject or population of subjects having the same type of tumor). Insome embodiments, an immune response is stimulated in a subject if thelevel of expression of one or more biomarkers of immune response (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers) is increased by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,90% or more relative to a control (e.g., a sample from the subject priorto the application of the nsPEF, or a sample from a control subject inwhich the nsPEF has not been applied).

For measuring one or more biomarkers of immune response, one or morebiological samples from the subject can be used. In some embodiments,the sample is from whole blood, plasma, serum, saliva, urine, stool,tears, any other bodily fluid, tissue samples (e.g., biopsy), andcellular extracts thereof (e.g., red blood cellular extract). In oneembodiment, the sample is a serum sample. The use of samples such asserum, saliva, and urine is well known in the art (see, e.g., Hashida etal., J. Clin. Lab. Anal., 11:267-86 (1997)). One skilled in the art willappreciate that samples such as serum samples can be diluted prior tothe analysis of biomarker levels. In some embodiments, the sample is abiopsy.

Biomarkers of Immune Response

In some embodiments, the biomarker of immune response is a white bloodcell, for example, but not limited to a lymphocyte. In some embodiments,the biomarker of immune response is a T cell. Examples of T cellsinclude, but are not limited to, CD4+ T cells and CD8+ T cells. In someembodiments, the biomarker of immune response is a cell that is positivefor one or more of the CD markers CD3, CD4, CD8, CD16, or CD56.

In some embodiments, the biomarker of immune response is a cytokine. Theterm “cytokine” includes any of a variety of polypeptides or proteinssecreted by immune cells that regulate a range of immune systemfunctions and encompasses small cytokines such as chemokines. In someembodiments, the biomarker of immune response is an inflammatorycytokine. In some embodiments, the biomarker of immune response is acytokine selected from GM-CSF, IL2, IL-4, IL-5, IL-6, IL-10, IL-12,IL-13, IL-15, interferon-γ, or TNF-α.

In some embodiments, the biomarker of immune response is a polypeptideor protein that is produced in an inflammatory or immune response. Insome embodiments, the biomarker of immune response is acute phaseprotein, including but not limited to C-reactive protein (CRP) or serumamyloid A.

In some embodiments, the biomarker of immune response is an antibody toa tumor cell surface marker. As used herein, the term “antibody”includes a population of immunoglobulin molecules, which can bepolyclonal or monoclonal and of any isotype, or an immunologicallyactive fragment of an immunoglobulin molecule. Such an immunologicallyactive fragment contains the heavy and light chain variable regions,which make up the portion of the antibody molecule that specificallybinds an antigen. For example, an immunologically active fragment of animmunoglobulin molecule known in the art as Fab, Fab′ or F(ab)₂ isincluded within the meaning of the term antibody. In some embodiments,the biomarker of immune response is an antibody to a cell surfacetumor-associated antigen (TAA). In some embodiments, the biomarker ofimmune response is an antibody to a tumor cell surface marker selectedfrom Human Epidermal Growth Factor Receptor 2 (HER2), Epidermal GrowthFactor Receptor (EGFR), Carbonic Anhydrase IX (CAIX), VascularEndothelial Growth Factor Receptor (VEGFR), VEGFR2, c-Met, prostate stemcell antigen (PSCA), Epithelial-Specific Cell Adhesion ActivationMolecule (EpCAM), carcinoembryonic antigen (CEA), or CA-125.

In some embodiments, stimulation of an immune response is measured bydetermining the level or concentration of one or more biomarkers ofimmune response (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkersof immune response) selected from the group consisting of a white bloodcell, a CD4⁺ T cell, a CD8+ T cell, a natural killer T cell, GM-CSF,IL2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, interferon-γ, TNF-α,C-reactive protein, serum amyloid A, and an antibody to a tumor cellsurface marker (e.g., a cell surface tumor-associated antigen (TAA),Human Epidermal Growth Factor Receptor 2 (HER2), Epidermal Growth FactorReceptor (EGFR), Carbonic Anhydrase IX (CAIX), Vascular EndothelialGrowth Factor Receptor (VEGFR), VEGFR2, c-Met, prostate stem cellantigen (PSCA), Epithelial-Specific Cell Adhesion Activation Molecule(EpCAM), carcinoembryonic antigen (CEA), or CA-125). One skilled in theart will recognize that other tumor cell surface markers are suitablefor use in the present invention.

Assays for Measuring Stimulation of Immune Response

Any of a variety of assays, techniques, and kits known in the art can beused to measure the stimulation of immune response in a sample. In someembodiments, a qualitative assay is used to determine whether theconcentration or level of the one or more biomarkers of immune responseis above a threshold level or is increased relative to a control. Insome embodiments, a quantitative assay is used to determine the relativeor absolute amount of the one or more biomarkers of immune response,e.g., for determining whether the concentration or level of the one ormore biomarkers of immune response is above a threshold level or isincreased relative to a control.

In some embodiments, the concentration or level of a biomarker of immuneresponse (e.g., a white blood cell, a cytokine, a protein produced in aninflammatory or immune response, or an antibody to a tumor cell surfacemarker) is measured by flow cytometry. Flow cytometry methods andinstrumentation are known in the art. Descriptions of instrumentationand methods can be found, e.g., in Introduction to Flow Cytometry: ALearning Guide (2000) Becton, Dickinson, and Company; McHugh, “FlowMicrosphere Immunoassay for the Quantitative and Simultaneous Detectionof Multiple Soluble Analytes,” Methods in Cell Biology 42, Part B(Academic Press, 1994).

In some embodiments, the concentration or level of a biomarker of immuneresponse (e.g., a white blood cell, a cytokine, a protein produced in aninflammatory or immune response, or an antibody to a tumor cell surfacemarker) is measured by phage display. For example, phage displaytechnology for expressing a recombinant antigen specific for an antibodybiomarker (e.g., an antibody to a tumor cell surface marker) can also beused to determine the presence or level of the antibody biomarker. Phageparticles expressing an antigen specific for, e.g., an antibody marker,can be anchored, if desired, to a multi-well plate using an antibodysuch as an anti-phage monoclonal antibody (Felici et al.,“Phage-Displayed Peptides as Tools for Characterization of Human Sera”in Abelson (Ed.), Methods in Enzymol., 267, San Diego: Academic Press,Inc. (1996)).

In some embodiments, the concentration or level of a biomarker of immuneresponse (e.g., a white blood cell, a cytokine, a protein produced in aninflammatory or immune response, or an antibody to a tumor cell surfacemarker) is measured by immunoassay. A variety of immunoassay techniques,including competitive and non-competitive immunoassays, can be used todetermine the concentration or level of the one or more biomarkers ofimmune response. See, e.g., Self and Cook, Curr. Opin. Biotechnol.,7:60-65 (1996)). The term immunoassay encompasses techniques including,without limitation, enzyme immunoassays (EIA) such as enzyme multipliedimmunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA),antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MACELISA), and microparticle enzyme immunoassay (META); capillaryelectrophoresis immunoassays (CEIA); radioimmunoassays (RIA);immunoradiometric assays (IRMA); fluorescence polarization immunoassays(FPIA); and chemiluminescence assays (CL). If desired, such immunoassayscan be automated. Immunoassays can also be used in conjunction withlaser-induced fluorescence (see, e.g., Schmalzing and Nashabeh,Electrophoresis, 18:2184-2193 (1997); Bao, J. Chromatogr. B. Biomed.Sci., 699:463-480 (1997)). Liposome immunoassays, such as flow-injectionliposome immunoassays and liposome immunosensors, are also suitable foruse in the present invention (see, e.g., Rongen et al., J. Immunol.Methods, 204:105-133 (1997)). In addition, nephelometry assays, in whichthe formation of protein/antibody complexes results in increased lightscatter that is converted to a peak rate signal as a function of themarker concentration, are suitable for use in the present invention.Nephelometry assays are commercially available from Beckman Coulter(Brea, Calif.; Kit #449430) and can be performed using a BehringNephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biol. Chem.,27:261-276 (1989)).

In some embodiments, antigen capture ELISA can be used to determine thepresence or level of one or more biomarkers of immune response in asample. For example, in an antigen capture ELISA, an antibody directedto a marker of interest is bound to a solid phase and sample is addedsuch that the marker is bound by the antibody. After unbound proteinsare removed by washing, the amount of bound marker can be quantitatedusing, e.g., a radioimmunoassay (see, e.g., Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988)).Sandwich ELISA can also be suitable for use in the present invention.For example, in a two-antibody sandwich assay, a first antibody is boundto a solid support, and the marker of interest is allowed to bind to thefirst antibody. The amount of the marker is quantitated by measuring theamount of a second antibody that binds the marker. The antibodies can beimmobilized onto a variety of solid supports, such as magnetic orchromatographic matrix particles, the surface of an assay plate (e.g.,microtiter wells), pieces of a solid substrate material or membrane(e.g., plastic, nylon, paper), and the like. An assay strip can beprepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

A radioimmunoassay using, for example, an iodine-125 (¹²⁵I) labeledsecondary antibody (Harlow and Lane, supra) is also suitable fordetermining the presence or level of one or more biomarkers of immuneresponse in a sample. A secondary antibody labeled with achemiluminescent marker can also be suitable for use in the presentinvention. A chemiluminescence assay using a chemiluminescent secondaryantibody is suitable for sensitive, non-radioactive detection of markerlevels. Such secondary antibodies can be obtained commercially fromvarious sources, e.g., Amersham Lifesciences, Inc. (Arlington Heights,Ill.).

Specific immunological binding of an antibody to one or more biomarkersof immune response can be detected directly or indirectly. Direct labelsinclude fluorescent or luminescent tags, metals, dyes, radionuclides,and the like, attached to the antibody. An antibody labeled withiodine-125 (¹²⁵I) can be used for determining the levels of one or moremarkers in a sample. A chemiluminescence assay using a chemiluminescentantibody specific for the marker is suitable for sensitive,non-radioactive detection of marker levels. An antibody labeled withfluorochrome is also suitable for determining the levels of one or moremarkers in a sample. Examples of fluorochromes include, withoutlimitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin,B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine.Secondary antibodies linked to fluorochromes can be obtainedcommercially, e.g., goat F(ab′)₂ anti-human IgG-FITC is available fromTago Immunologicals (Burlingame, Calif.) or its current incarnation,such as BioSource, Invitrogen, or Life Technologies.

Indirect labels include various enzymes well-known in the art, such ashorseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, urease, and the like. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable at 450 nm. Analkaline phosphatase detection system can be used with the chromogenicsubstrate p-nitrophenyl phosphate, for example, which yields a solubleproduct readily detectable at 405 nm. Similarly, a β-galactosidasedetection system can be used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a solubleproduct detectable at 410 nm. An urease detection system can be usedwith a substrate such as urea-bromocresol purple (Sigma Immunochemicals;St. Louis, Mo.). A useful secondary antibody linked to an enzyme can beobtained from a number of commercial sources, e.g., goat F(ab′)₂anti-human IgG-alkaline phosphatase can be purchased from JacksonImmunoResearch (West Grove, Pa.).

A signal from the direct or indirect label can be analyzed, for example,using a spectrophotometer to detect color from a chromogenic substrate;a radiation counter to detect radiation such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. For detection ofenzyme-linked antibodies, a quantitative analysis of the amount ofmarker levels can be made using a spectrophotometer such as an EMAXMicroplate Reader (Molecular Devices; Menlo Park, Calif.) in accordancewith the manufacturer's instructions. If desired, the assays of thepresent invention can be automated or performed robotically, and thesignal from multiple samples can be detected simultaneously.

Quantitative Western blotting can also be used to detect or determinethe presence or level of one or more biomarkers of immune response in asample. Western blots can be quantitated by well-known methods such asscanning densitometry or phosphorimaging. As a non-limiting example,protein samples are electrophoresed on 10% SDS-PAGE Laemmli gels.Primary murine monoclonal antibodies are reacted with the blot, andantibody binding can be confirmed to be linear using a preliminary slotblot experiment. Goat anti-mouse horseradish peroxidase-coupledantibodies (BioRad, Hercules, Calif.) are used as the secondaryantibody, and signal detection performed using chemiluminescence, forexample, with the Renaissance chemiluminescence kit (New EnglandNuclear; Boston, Mass.) according to the manufacturer's instructions.Autoradiographs of the blots are analyzed using a scanning densitometer(Molecular Dynamics; Sunnyvale, Calif.) and normalized to a positivecontrol. Values are reported, for example, as a ratio between the actualvalue to the positive control (densitometric index). Such methods arewell known in the art as described, for example, in Parra et al., J.Vasc. Surg., 28:669-675 (1998).

Alternatively, a variety of immunohistochemical assay techniques can beused to determine the presence or level of one or more biomarkers ofimmune response in a sample. The term immunohistochemical assayencompasses techniques that utilize the visual detection of fluorescentdyes or enzymes coupled (i.e., conjugated) to antibodies that react withthe marker of interest using fluorescent microscopy or light microscopyand includes, without limitation, direct fluorescent antibody assay,indirect fluorescent antibody (IFA) assay, anticomplementimmunofluorescence, avidin-biotin immunofluorescence, andimmunoperoxidase assays. An IFA assay, for example, is useful fordetermining whether a sample is positive for a biomarker, the level ofbiomarker in a sample, and/or a biomarker's staining pattern. Theconcentration of the biomarker in a sample can be quantitated, e.g.,through endpoint titration or through measuring the visual intensity offluorescence compared to a known reference standard.

In some embodiments, the concentration or level of a biomarker of immuneresponse (e.g., a white blood cell, a cytokine, a protein produced in aninflammatory or immune response, or an antibody to a tumor cell surfacemarker) is measured by detecting or quantifying the amount of thepurified marker. Purification of the marker can be achieved, forexample, by high pressure liquid chromatography (HPLC), alone or incombination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, tandemMS, etc.). Qualitative or quantitative detection of a marker of interestcan also be determined by well-known methods including, withoutlimitation, Bradford assays, Coomassie blue staining, silver staining,assays for radiolabeled protein, and mass spectrometry.

In some embodiments, the concentration or level of a biomarker of immuneresponse (e.g., a white blood cell, a cytokine, a protein produced in aninflammatory or immune response, or an antibody to a tumor cell surfacemarker) is measured by analysis of marker mRNA levels using routinetechniques such as Northern analysis, reverse-transcriptase polymerasechain reaction (RT-PCR), or any other methods based on hybridization toa nucleic acid sequence that is complementary to a portion of the markercoding sequence (e.g., slot blot hybridization) are also within thescope of the present invention. Applicable PCR amplification techniquesare described in, e.g., Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, Inc. New York (1999), Chapter 7 andSupplement 47; Theophilus et al., “PCR Mutation Detection Protocols,”Humana Press, (2002); and Innis et al., PCR Protocols, San Diego,Academic Press, Inc. (1990). General nucleic acid hybridization methodsare described in Anderson, “Nucleic Acid Hybridization,” BIOS ScientificPublishers, 1999. Amplification or hybridization of a plurality oftranscribed nucleic acid sequences (e.g., mRNA or cDNA) can also beperformed from mRNA or cDNA sequences arranged in a microarray.Microarray methods are generally described in Hardiman, “MicroarraysMethods and Applications: Nuts & Bolts,” DNA Press, 2003; and Baldi etal., “DNA Microarrays and Gene Expression: From Experiments to DataAnalysis and Modeling,” Cambridge University Press, 2002.

For the detection of a plurality of biomarkers (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10 or more biomarkers), the analysis of the biomarkers may becarried out separately or simultaneously with one test sample. Forseparate or sequential assay of markers, suitable apparatuses includeclinical laboratory analyzers such as the ElecSys (Roche), the AxSym(Abbott), the Access (Beckman), the ADVIA®, the CENTAUR® (Bayer), andthe NICHOLS ADVANTAGE® (Nichols Institute) immunoassay systems.Preferred apparatuses or protein chips perform simultaneous assays of aplurality of markers on a single surface. Particularly useful physicalformats comprise surfaces having a plurality of discrete, addressablelocations for the detection of a plurality of different markers. Suchformats include protein microarrays, or “protein chips” (see, e.g., Nget al., J. Cell Mol. Med., 6:329-340 (2002)) and certain capillarydevices (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments, eachdiscrete surface location may comprise antibodies to immobilize one ormore markers for detection at each location. Surfaces may alternativelycomprise one or more discrete particles (e.g., microparticles ornanoparticles) immobilized at discrete locations of a surface, where themicroparticles comprise antibodies to immobilize one or more markers fordetection. Yet another suitable format for performing simultaneousassays of a plurality of markers is the Luminex MultiAnalyte Profiling(xMAP) technology, previously known as FlowMetrix and LabMAP (Elshal andMcCoy, 2006), a multiplex bead-based flow cytometric assay that utilizespolystyrene beads that are internally dyed with different intensities ofred and infrared fluorophores. The beads can be bound by various capturereagents such as antibodies, oligonucleotides, and peptides, thereforefacilitating the quantification of various biomarkers such as proteins,ligands, DNA and RNA (Fulton et al., 1997; Kingsmore, 2006; Nolan andMandy, 2006, Vignali, 2000; Ray et al., 2005).

Several biomarkers of interest may be combined into one test forefficient processing of a multiple of samples. In addition, one skilledin the art would recognize the value of testing multiple samples (e.g.,at successive time points, etc.) from the same subject. For example,samples can be collected daily, weekly, monthly, or at other intervalsfrom a subject and tested. Such testing of serial samples can allow theidentification of changes in marker levels over time. Increases ordecreases in marker levels, as well as the absence of change in markerlevels, can also provide useful information to measure and/or quantifystimulation of immune response.

An immune booster can be administered in order to help create conditionsfor stimulating the immune systems. Natural and synthetic supplementsthat have been thought to be immune boosters at one time or anotherinclude flaxseed oil, sodium ascorbate (i.e., vitamin C), calciumascorbate, magnesium ascorbate, potassium ascorbate, zinc ascorbate,manganese ascorbate, and chromium ascorbate, and cayenne pepper(capsicum), astragalus, Echinacea, esberitox, olive leaf extract,sambucus (elderberry), umcka, (umckaloabo), and zinc.

It is understood that the various embodiments described herein are byway of example only, and are not intended to limit the scope of theinvention. For example, many of the materials and structures describedherein may be substituted with other materials and structures withoutdeviating from the spirit of the invention. The present invention asclaimed may therefore include variations from the particular examplesand preferred embodiments described herein, as will be apparent to oneof skill in the art. It is understood that various theories as to whythe invention works are not intended to be limiting.

The above description is illustrative and is not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of the disclosure. The scope of the invention should,therefore, be determined not with reference to the above description,but instead should be determined with reference to the pending claimsalong with their full scope or equivalents.

As noted previously, all measurements, dimensions, and materialsprovided herein within the specification or within the figures are byway of example only.

A recitation of “a,” “an,” or “the” is intended to mean “one or more”unless specifically indicated to the contrary. Reference to a “first”component does not necessarily require that a second component beprovided. Moreover reference to a “first” or a “second” component doesnot limit the referenced component to a particular location unlessexpressly stated.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedmay be different from the actual publication dates, which may need to beindependently confirmed.

What is claimed is:
 1. A method for stimulating an immune response to adisease in a subject, the method comprising: determining a targettreatment energy based on a size and a type of a tumor in the subject;selecting or allowing selection of a number of pulses, an amplitude ofat least 10 kilovolts per centimeter, or a pulse length of between 0.1nanoseconds and 1000 nanoseconds for sub-microsecond pulsed electricfields based on the determined target treatment energy; and applying tothe tumor, using a plurality of electrodes positioned in or around thetumor or a portion thereof, the sub-microsecond pulsed electric fieldshaving the selected number of pulses, the selected amplitude, or theselected pulse length.
 2. The method of claim 1 further comprising:adjusting the target treatment energy based on a comparison of ameasured immune response biomarker before the applying and a measuredimmune response biomarker after the applying in a sample of the subject;determining a new number of pulses, a new amplitude, or a new pulselength based on the adjusted target treatment energy; and treating thetumor again by: applying to the tumor, using the plurality ofelectrodes, sub-microsecond pulsed electric fields based on the newdetermined number of pulses, the new determined amplitude, or the newdetermined pulse length.
 3. The method of claim 1, wherein the method isused in combination with introducing cluster of differentiation 47(CD47)-blocking antibodies into the subject, the CD47-blockingantibodies neutralizing CD47 on surface membranes of tumor cells of thetumor.
 4. The method of claim 3, wherein the introducing ofCD47-blocking antibodies occurs before the applying of thesub-microsecond pulsed electric fields.
 5. The method of claim 1,wherein the method is used in combination with injecting doxorubicininto the subject.
 6. The method of claim 1, wherein the method is usedin combination with injecting cytotoxic T-lymphocyte antigen 4(CTLA-4)-blocking antibodies into the subject.
 7. The method of claim 1,wherein the method is used in combination with injecting programmeddeath 1 (PD-1)-blocking antibodies into the subject.
 8. The method ofclaim 1, the method further comprising obtaining a measurement of animmune response biomarker in a sample of the subject, wherein themeasurement is based on a concentration or level of white blood cells inthe sample.
 9. The method of claim 8, wherein the measured white bloodcells include cluster of differentiation 4+(CD4+) or cluster ofdifferentiation 8+(CD8+) T lymphocytes.
 10. The method of claim 2,wherein the gauged immune response biomarker is based on: measuring aconcentration or level in the sample of a member selected from a groupconsisting of white blood cells, inflammatory cytokines, C-reactiveproteins, and antibodies of cancer cell markers.
 11. The method of claim1, wherein the tumor is selected among multiple tumors in the subjectbased on an accessibility of locations and measured sizes of themultiple tumors.
 12. The method of claim 11 wherein the accessibility ofeach tumor is determined by identifying pathways to opposed sides of arespective tumor that can be contacted by electrodes through pierceablepaths in the subject.
 13. The method of claim 1, wherein the methodreduces metastasis of the disease in the subject.
 14. The method ofclaim 1, wherein the tumor is cancerous, precancerous, or non-cancerous.15. The method of claim 1, wherein one or more of the number of pulses,the amplitude, or the pulse length are inputted though a user interface.16. The method of claim 1, the method comprises obtaining a measurementof an immune response biomarker, wherein the measurement is automated.17. The method of claim 1, wherein the disease is one or more of thefollowing cancers: digestive, gastrointestinal, colorectal,gastrointestinal stromal, colon, rectal, anal, bile duct, smallintestine, esophageal, breast, lung, gallbladder, liver, pancreatic,appendix, prostate, ovarian, renal, cancer of the central nervoussystem, skin, lymphomas, gliomas, choriocarcinomas, head and neck,osteogenic sarcomas, or blood cancers.
 18. The method of claim 1, themethod further comprising: detecting calreticulin on surface membranesof cells of the tumor after the applying.
 19. The method of claim 1,wherein the selected number of pulses is greater than
 50. 20. Amachine-readable tangible medium storing instructions for causing one ormore machines to execute operations for: determining a target treatmentenergy based on a size and a type of a tumor in a subject; selecting anumber of pulses, an amplitude of at least 10 kilovolts per centimeter,or a pulse length of between 0.1 nanoseconds and 1000 nanoseconds forsub-microsecond pulsed electric fields based on the determined targettreatment energy; and applying to the tumor, through a set ofelectrodes, the sub-microsecond pulsed electric fields having theselected number of pulses, the selected amplitude, or the selected pulselength.
 21. The medium of claim 20, further comprising instructions for:selecting a repetition frequency of the sub-microsecond pulsed electricfields based at least on the determined size and type of the tumor. 22.The medium of claim 20, wherein the tumor is cancerous, precancerous, ornon-cancerous.